Our project is examining phloem gene expression changes in response to CLas infection in HLB-susceptible sweet orange and HLB-resistant Poncirus and Carrizo (a sweet orange – Poncirus cross). We are using a recently developed methodology for woody crops that allows gene expression profiling of phloem tissues. The method leverages a translating ribosome affinity purification strategy (called TRAP) to isolate and characterize translating mRNAs from phloem specific tissues. Our approach is unlike other gene expression profiling methods in that it only samples gene transcripts that are actively being transcribed into proteins and is thus a better representation of active cellular processes than total cellular mRNA. Sweet orange, and HLB-resistant Poncirus and Carrizo (sweet orange x Poncirus) will be transformed to express the tagged ribosomal proteins under the control of characterized phloem-specific promoters; tagged ribosomal proteins under control of the nearly ubiquitous CaMV 35S promoter will be used as a control. Transgenic plants will be exposed to CLas+ or CLas- ACP and leaves sampled 1, 2, 4, 8, and 12 weeks later. Ribosome-associated mRNA will be sequenced an(he 3nd quarter of the third year of our grant, the Stover lab continues to grow the last few transgenic citrus in preparation for shipping them to the Rogers lab, which should happen early in the 4th quarter. The Rogers lab has continued small-scale no-choice psyllid inoculation experiments on the many lines they have received from the Stover lab and has begun RNA extractions. ARS facilities are still at a maximum of 25% occupancy due to the COVID-19 pandemic; we are teleworking the remaining time. This continues to slow down progress on grant milestones. We are very much hoping to be allowed to move to the next phases of reopening soon, which will allow for much more rapid progress. In addition, Dr. Rogers is advertising for a new post-doc to work on finishing this project.